A colorimetric assay using the basic azo dye Janus green has been developed to assess cell numbers in anchorage-dependent cell cultures, with special regard to the enumeration of osteoblastic cells. Therefore, cells are fixed in ethanol and stained with a 0.2% solution of Janus green for 3 min, followed by a destaining step of 1 min in tap water. The addition of diluted hydrochloric acid easily and immediately leads to dye elution from stained cell layers into the acidic supernatant which consequently is transferred into 96-well plates and read on a microplate reader at 595 nm. Working under standardized conditions, Janus green uptake in several cell lines is shown to be linearly correlated with cell numbers over a broad range of cell densities, in MC3T3-E1 cells from about 3% up to more than 300% of confluency. Absolute sensitivity of the assay allows detection of less than 1000 cells/cm². In comparison to many other colorimetric assays, the Janus green technique is simple to perform, fast, precise, stable, cheap, and well suited for processing large quantities of samples. Moreover, it is applicable to any culture formate and size, from irregular formed carriers up to 96-multiwell plates.